CN114026217A

CN114026217A - Cell recovery method and cell culture apparatus

Info

Publication number: CN114026217A
Application number: CN202080044142.1A
Authority: CN
Inventors: 堀井大地; 竹内晴纪; 须田佳雅; 占部雄士
Original assignee: Sinfonia Technology Co Ltd
Current assignee: Sinfonia Technology Co Ltd
Priority date: 2019-06-20
Filing date: 2020-06-15
Publication date: 2022-02-08
Also published as: TW202100740A; US20220356437A1; JP2021000007A; JP7428866B2; WO2020255930A1; KR20220024054A; EP3988639A4; EP3988639A1

Abstract

The present invention provides a cell recovery method without a centrifuge to suppress damage to cells. When the cells cultured in at least one vessel containing a liquid medium and adhering to the inner surface of the vessel are recovered, a medium discharging step (step S11) of discharging the medium from the vessel is performed; a stripping liquid supply step (step S12) of supplying a stripping liquid for stripping cells from the inner surface of the container to the container after discharging the culture medium; a stripping liquid discharging step (step S14) of discharging the stripping liquid from the container before the cells are completely stripped from the inner surface of the container; a waiting step (step S15) of waiting until the cells are peeled off by the residual peeling liquid after the peeling liquid is discharged; and a recovery liquid supply step (step S16) of supplying a recovery liquid for recovering cells to the container after the waiting step is completed.

Description

Cell recovery method and cell culture apparatus

Technical Field

The present invention relates to a cell recovery method for recovering cells cultured in a vessel containing a liquid medium and adhering to the inner surface of the vessel, and a cell culture apparatus capable of performing the cell recovery method.

Background

When culturing cells in a vessel containing a liquid medium, the cells in the vessel need to be recovered at the time of subculture (subculture) in another vessel during the culture or at the time of harvesting the cells after the completion of the culture. Conventionally, such cell recovery operation is carried out in the following manner. That is, after the culture medium in the container is discharged, a stripping liquid is supplied to the container, and the cells adhered to the inner surface of the container are stripped by the action of the stripping liquid. Next, the cell-containing stripping solution is transferred to a centrifuge tube, and the cells in the centrifuge tube are separated from the stripping solution by a centrifuge, thereby recovering the cells.

In recent years, development of a cell culture apparatus for automatically culturing iPS cells and ES cells has been promoted. However, when a centrifuge is incorporated into such a cell culture apparatus, there is a problem in that the apparatus becomes large and the cost increases. In view of this, the cell culture apparatus described in patent document 1 employs a cell recovery method that does not require a centrifuge. Specifically, after the supply of the stripping solution, when the adhesion of the cells is weakened, the stripping solution is discharged, and then the culture medium is supplied to the vessel. The cells are at this time peeled from the inner surface of the container by the force of the flowing medium. This eliminates the need to separate the cells from the stripping solution, thereby eliminating the need for a centrifuge.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2017-6058

Disclosure of Invention

Problems to be solved by the invention

However, in the cell recovery method described in patent document 1, the cells having weak adhesion are peeled from the inner surface of the container by the force of the flowing medium. Therefore, an excessive force is applied to the cells, which may damage the cells. In addition, in patent document 1, the cells are exfoliated before the action of the exfoliating solution is sufficiently exerted on the cells. Thus, the cells that are exfoliated are present in the form of agglomerates. Thus, after the cells are recovered, the cell agglomerates are destroyed by passing the cell-containing medium (cell suspension) through a tube pump. In this step, the cells may also be damaged by being handled by the tube pump.

The present invention takes the above into consideration and provides some embodiments of a cell recovery method that does not require a centrifuge and can reduce damage to cells.

Means for solving the problems

According to an embodiment of the present invention, there is provided a cell recovery method for recovering cells cultured in at least one vessel containing a liquid medium and adhered to an inner surface of the vessel, the method including performing: a medium discharging step of discharging a liquid medium from a container; a stripping solution supply step of supplying a stripping solution for stripping cells from the inner surface of the container to the container after the liquid medium is discharged; a stripping liquid discharging step of discharging the stripping liquid from the container before completely stripping the cells from the inner surface of the container; a waiting step of waiting until the cells are peeled off by the residual peeling liquid after the peeling liquid is discharged; and a recovery liquid supply step of supplying a recovery liquid for recovering cells to the container after the waiting step is completed.

In the cell recovery method according to the present invention, after the stripping liquid is discharged, after waiting until the cells are stripped by the residual stripping liquid, the recovery liquid is supplied. Therefore, it is not necessary to separate the cells from the stripping solution, and it is possible to eliminate the need for a centrifuge. In addition, since the process is continued until the cells are separated by the residual stripping solution, it is not necessary to forcibly separate the cells adhering to the inner surface of the container by the flowing recovery solution. In addition, since the cells are sufficiently separated from each other by the residual stripping solution, it is not necessary to decompose the cell aggregates by a tube pump or the like. Therefore, according to the present invention, it is possible to suppress damage to cells while eliminating the need for a centrifuge.

According to another embodiment of the present invention, there is provided a cell culture apparatus configured to perform the above cell recovery method, including: a medium supply/discharge device configured to supply and discharge a liquid medium to and from the vessel; a stripping liquid supply/discharge device configured to supply and discharge a stripping liquid to and from the container, a recovery liquid supply/discharge device configured to supply and discharge a recovery liquid to and from the container; and a controller, wherein the cell recovery method is performed by the controller controlling operations of the medium supply/discharge device, the stripping liquid supply/discharge device, and the recovery liquid supply/discharge device.

According to the cell culture apparatus of the present invention, the above-described cell recovery method can be automatically performed without human intervention.

In the cell culture apparatus according to the present invention, the at least one container may include at least two containers, the at least two containers may be connected by a connection path, and the recovery liquid containing the cells in one of the at least two containers may be transferred to the other of the at least two containers by supplying a gas to one of the at least two containers after the cell recovery method is performed in the one of the at least two containers.

According to such a configuration, during subculture in which a cell suspension (a recovery liquid containing cells) is transferred from one of the at least two vessels to the other of the at least two vessels, the cell suspension can be transferred without passing through a tube pump or the like. Therefore, it is possible to suppress damage to cells.

Drawings

FIG. 1 is a schematic front view showing the configuration of a cell culture apparatus according to an embodiment of the present invention.

FIG. 2 is a front view showing the configuration of the incubator.

FIG. 3 is a view showing a culture circuit formed inside the cell culture apparatus.

FIG. 4 is a flowchart showing a series of procedures for subculture.

FIG. 5 is a schematic view showing a cell recovery operation.

Detailed Description

Embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

(arrangement of cell culture apparatus)

As shown in fig. 1, the cell culture apparatus 1 according to the present embodiment includes a refrigerating reservoir 2, a heater 3, an incubator 4, and a controller 5. The cell culture apparatus 1 is an apparatus that cultures cells based on data input and stored in the controller 5. In the following description, the front-rear direction is defined as a direction perpendicular to the paper surface in fig. 1.

The refrigerating reservoir 2 and the heater 3 are an outer casing in which a rack for arranging containers containing media or reagents is formed. Although not shown, the front surfaces of the refrigerating reservoir 2 and the heater 3 are provided with doors that can open and close openings formed in the front surface of the housing. The refrigerating reservoir 2 is provided with a cooling mechanism (not shown), and the inside temperature of the cooling mechanism is maintained at an arbitrary temperature lower than the room temperature. The heater 3 is provided in the incubator 4, and the temperature in the heater 3 is substantially equal to the temperature in the incubator 4. Further, the tube is connected to a container provided in the refrigerating reservoir 2 and a container provided in the heater 3 so that the liquid in the containers can flow out through the tube. The liquid in the container can be discharged by a pump 102 described later. Examples of containers include bottles, bags, and the like.

As shown in fig. 1 and 2, the incubator 4 includes a first chamber 11 including a first opening/closing device 21 on a front surface thereof; and a second chamber 12 including a second on/off switch 22 on a front surface thereof and an environment conditioner 13. The first and second opening/

closing devices

21 and 22 and the wall surfaces of the first and

second chambers

11 and 12 are made of a heat insulating material. Therefore, in a state where the first and second opening/

closing devices

21 and 22 are closed, the temperatures in the first and

second chambers

11 and 12 are maintained. As shown in fig. 2, a sealed container 50 is installed in the first chamber 11, and a sealed container 60 is installed in the second chamber 12. The inside of the closed

containers

50 and 60 is sterile. Examples of containers include flasks, multi-layered containers, bags, and the like. In addition, in the present embodiment, the closed

vessels

50 and 60 are made of a material having CO₂A permeable material. However, the

containment vessels

50 and 60 may be constructed so as not to allow CO₂Made of the same material. The volume of the closed casing 60 is larger than that of the closed casing 50. This is because when cells cultured in closed vessel 50 with a high concentration are transferred into closed vessel 60 and further cultured in closed vessel 60, the amount of the medium contained in closed vessel 60 needs to be larger than the amount of the medium contained in closed vessel 50. However, it is not essential that the volume of the closed vessel 60 be larger than the volume of the closed vessel 50. The volume of containment vessel 60 may be less than or equal to the volume of containment vessel 50. The dotted line portion in fig. 2 indicates that some components are disposed in the first chamber 11 and the second chamber 12.

The environmental conditioner 13 includes a built-in heating device and CO₂Supply means and may be based on a controller 5The emitted signals regulate the temperature and CO in the first and

second chambers

11 and 12₂And (4) concentration. In addition, for detecting temperature and CO₂A sensor 23 of concentration is arranged in the first chamber 11 and the second chamber 12. Information detected by the sensor 23 is output to the controller 5. Means for adjusting other internal environments may be installed in the environment conditioner 13. In this case, the sensor 23 is a sensor that can detect other internal environments as well.

Further, as shown in FIG. 2, the incubator 4 includes a connection path 30 for connecting the closed vessel 50 and the closed vessel 60 to each other, and a driver 40 for moving the cells between the closed vessel 50 and the closed vessel 60 through the connection path 30. The connection path 30 includes a tube 71 and a stirrer 32, and the inside thereof is maintained in a sterile state. The stirrer 32 is connected to the

closed vessels

50 and 60 through a pipe 71. Further, the driver 40 includes a pump 101 and a gas tank 33 connected to the pump 101 and containing gas therein. The gas tank 33 is connected to the

hermetic containers

50 and 60 through a pipe 72. The gas contained in the gas tank 33 may be, for example, CO₂And may be another gas or a mixed gas composed of a plurality of types of gases. In fig. 2, some of the pipes and pumps are omitted from illustration.

(culture circuit)

FIG. 3 shows a closed culture loop 70 that enables cell culture in

closed vessels

50 and 60 while maintaining sterility in

closed vessels

50 and 60. The culture circuit 70 is provided with a culture medium container 34, a stripping liquid container 35, a waste liquid container 36, and the like, in addition to the

closed vessels

50 and 60, the agitator 32, and the gas tank 33, which have been described above. These components are connected by pipes 71 to 74. Hereinafter, a detailed description will be given.

The stirrer 32 is connected to the

closed vessels

50 and 60 through a pipe 71. A valve V1 is provided in the pipe 71 between the closed casing 50 and the agitator 32, and a valve V2 is provided in the pipe 71 between the closed casing 60 and the agitator 32. Further, the gas tank 33 is connected to the

closed vessels

50 and 60 through a pipe 72. A valve V3 is provided in the pipe 72 between the closed casing 50 and the gas tank 33, and a valve V4 is provided in the pipe 72 between the closed casing 60 and the gas tank 33.

The medium container 34 and the peeling liquid container 35 are provided inside the heater 3. The medium vessel 34 contains a liquid medium for culturing cells. The stripping solution container 35 contains a stripping solution for stripping cells adhered to the inner surfaces of the

closed containers

50 and 60. Although not shown, medium container 34 is connected to a medium pot provided in refrigerated storage 2 via a pipe, and medium is suitably supplied from the medium pot to medium container 34. Likewise, the stripping liquid container 35 is connected to a stripping liquid tank provided inside the refrigerating reservoir 2 through a pipe, and the culture medium is appropriately supplied from the stripping liquid tank to the stripping liquid container 35.

The medium vessel 34 and the stripping solution vessel 35 are connected to the

closed vessels

50 and 60 and the stirrer 32 through a pipe 73. A pump 102 is provided in a portion of the pipe 73 between the medium container 34 and the stripping liquid container 35 and the

closed vessels

50 and 60 and the agitator 32. Valves V5 to V9 are provided on the pipe 73. Valve V5 is provided between medium reservoir 34 and pump 102 to switch the state of medium supply from medium reservoir 34. A valve V6 is provided between the stripping liquid container 35 and the pump 102 to switch the supply state of the stripping liquid from the stripping liquid container 35. A valve V7 is provided between the closed vessel 50 and the pump 102 to switch the supply state of the culture medium or the stripping liquid to the closed vessel 50. A valve V8 is provided between the closed vessel 60 and the pump 102 to switch the supply state of the culture medium or the stripping liquid to the closed vessel 60. A valve V9 is provided between the agitator 32 and the pump 102 to switch the supply state of the culture medium or the stripping liquid to the agitator 32.

The waste liquid container 36 is a container for discharging waste liquid from the

closed containers

50 and 60 and the agitator 32. The waste liquid container 36 is constituted by a deaerator 37 communicating with the outside air. The gas in the waste liquid container 36 is released into the atmosphere through the deaerator 37. A check valve, a filter, etc. may be installed on the deaerator 37, if necessary.

The waste liquid container 36 is connected to the

closed containers

50 and 60 and the agitator 32 through a pipe 74. Valves V10 to V12 are provided on the pipe 74. A valve V10 is provided between the closed casing 50 and the pump 103 to switch the discharge state of the waste liquid from the closed casing 50. A valve V11 is provided between the closed casing 60 and the pump 103 to switch the discharge state of the waste liquid from the closed casing 60. A valve V12 is provided between the agitator 32 and the pump 103 to switch the discharge state of the waste liquid from the agitator 32. Pump 103 may be omitted if the waste fluid from

closed containers

50 and 60 and agitator 32 reach waste fluid container 36 under the force of gravity.

(cell recovery operation)

As an example of the cell recovery method according to the present invention, the operation at the time of subculture in which the cells cultured in the closed vessel 50 are transferred into the closed vessel 60 will be described with reference to FIGS. 4 and 5. FIG. 4 is a flowchart showing a series of procedures for subculture, and FIG. 5 is a schematic diagram showing a cell recovery operation. In the present embodiment, the controller 5 automatically controls the driving of the pumps 101 to 103 and the opening/closing of the valves V1 to V12 to automatically perform subculture. Various conditions related to the culture and subculture are input to the controller 5 in advance by the operator. At the start of subculture, it is assumed that all the valves V1 to V12 are closed and the inside of the culture circuit 70 is maintained in a sterile state.

When subculturing is performed from closed vessel 50 to closed vessel 60, first, as shown in a of FIG. 5, medium M is discharged from closed vessel 50 (step S11). Specifically, the controller 5 opens the valve V10 and drives the pump 103 to discharge the medium M in the closed vessel 50 into the waste liquid vessel 36 through the pipe 74. At this time, the cells C adhere to the inner surface of the closed vessel 50. Therefore, the cells C are not discharged together with the medium M. When the medium M in the closed vessel 50 is discharged, the controller 5 closes the valve V10 and stops the pump 103.

Next, as shown in b of fig. 5, the peeling liquid L is supplied to the closed casing 50 (step S12). Specifically, the controller 5 opens the valves V6 and V7 and drives the pump 102 to supply the stripping liquid L in the stripping liquid container 35 to the closed container 50 through the pipe 73. When a predetermined amount of the peeling liquid L is supplied to the closed vessel 50, the controller 5 closes the valves V6 and V7 and stops the pump 102.

After the supply of the peeling liquid L to the hermetic container 50 is completed, the process waits for a first predetermined time in this state (step S13). The first predetermined time is a time until the cells C adhered to the inner surface of the closed vessel 50 enter a state immediately before being completely peeled by the chemical action of the peeling liquid L. The first predetermined time is appropriately determined depending on the type of the cells C and the type of the peeling liquid L, and may be, for example, about 2 to 3 minutes.

When the first predetermined time has elapsed (YES in step S13), the peeling liquid L is discharged from the sealed container 50 as shown in c in FIG. 5 (step S14). Specifically, the controller 5 opens the valve V10 and drives the pump 103 to discharge the stripping liquid L in the closed vessel 50 into the waste liquid vessel 36 through the pipe 74. At this time, the cells C adhere to the inner surface of the closed vessel 50. Therefore, the cells C are not discharged together with the stripping solution L. If some cells C are exfoliated before the exfoliating liquid L is discharged, they may be discharged together with the exfoliating liquid L. However, there were very few, if any, such cells C. After the peeling liquid L in the closed vessel 50 is discharged, the controller 5 closes the valve V10 and stops the pump 103.

After the peeling liquid L is discharged from the hermetic container 50, the process waits for a second predetermined time in this state (step S15). In step S14, substantially the entire amount of peeling liquid L is drained from the hermetic container 50. However, in reality, as shown in d of fig. 5, the residual stripping solution L adheres to the inner surface of the closed vessel 50 and the cells C due to surface tension. The second predetermined time is a time until the cells C held in the state just before being completely peeled by step S13 are completely peeled by the chemical action of the residual peeling liquid L. The second predetermined time is appropriately determined depending on the type of the cells C and the type of the peeling liquid L, and is, for example, about 2 to 3 minutes.

When the second predetermined time has elapsed (YES in step S15), as shown in e in FIG. 5 (step S16), a fresh medium M is supplied to the closed vessel 50. Specifically, controller 5 supplies medium M in medium vessel 34 to closed vessel 50 through pipe 73 by opening valves V5 and V7 and driving pump 102. Due to the step S15, the cells C adhered to the inner surface of the hermetic container 50 are completely peeled off, and the cells C are in a disintegrated state, not in an agglomerated state. Thus, when the liquid medium M is supplied, the cells C in the closed vessel 50 are mixed with the medium M to form the cell suspension S. Since the amount of the remaining stripping solution L is small, the remaining stripping solution L is sufficiently diluted by the supplied medium M, and does not cause a problem in the subsequent steps. After supplying a predetermined amount of medium M to the closed vessel 50, the controller 5 closes the valves V5 and V7 and stops the pump 102.

Next, the cell suspension S in the closed vessel 50 is moved to the agitator 32 (step S17). Specifically, controller 5 opens valves V1 and V3 and drives pump 101 to pump CO in gas tank 33₂The mixture is fed into the closed vessel 50 through the pipe 72. As a result, the cell suspension S in the closed vessel 50 moves to the agitator 32 through the tube 71. After the cell suspension S moves to the agitator 32, the controller 5 closes the valves V1 and V3 and stops the pump 101.

Subsequently, the concentration of the cell suspension S in the agitator 32 is adjusted (step S18). Specifically, a small amount of the cell suspension S, the concentration of which is made uniform by being stirred by the stirrer 32, is brought to a cell counter (not shown) where the concentration of the cell suspension S is measured. Based on this measurement, the controller 5 calculates the amount of additional medium M required to bring the cell suspension S to a predetermined concentration. Then, the controller 5 opens the valves V5 and V9, and drives the pump 102 to supply a predetermined amount of medium M from the medium container 34 to the agitator 32. Therefore, the concentration of the cell suspension S contained in the agitator 32 can be adjusted to a predetermined level. After supplying a predetermined amount of the medium M to the agitator 32, the controller 5 closes the valves V5 and V9 and stops the pump 102.

Finally, the concentration-adjusted cell suspension S in the agitator 32 is moved to a new closed vessel 60 (step S19). Specifically, the controller 5 opens the valves V1 to V3 and drives the pump 101 to pump CO in the gas tank 33₂The mixture is fed into the stirrer 32 through the closed vessel 50. As a result, the cell suspension S in the stirrer 32 is transferred to the closed vessel 60 through the pipe 71. After the cell suspension S is moved into the closed vessel 60, the controller 5 closes the valves V1 to V3 and stops the pump 101. Thus, subculture was completed. Subculture can be repeated by replacing

closed vessels

50 and 60 with new closed vessels.

When the

hermetic containers

50 and 60 are replaced with new ones, the pipes, the stirrer 32, etc. connected to the

hermetic containers

50 and 60 may be replaced with new ones, or the insides of the pipes and the stirrer 32, etc. may be replaced with new ones, or may be reused after cleaning the insides thereof. When the

closed vessels

50 and 60 are replaced, the

closed vessels

50 and 60 are removed from the tubes while maintaining sterility within the culture loop 70, and additional new closed vessels are connected to the tubes while maintaining sterility within the additional new closed vessels. In this case, for example, a welding machine such as BioWelder (manufactured by Sartorius Stedim, japan) or an OPTA sterile connector (manufactured by Sartorius Stedim, japan) may be used.

Steps S11 to S16 in the above subculture operation correspond to the cell recovery method according to the present invention. Specifically, step S11 corresponds to the culture medium discharging step of the present invention, step S12 corresponds to the stripping liquid supplying step of the present invention, step S14 corresponds to the stripping liquid discharging step of the present invention, step S15 corresponds to the waiting step of the present invention, and step S16 corresponds to the recovering liquid supplying step of the present invention. Further, the medium M is used as a recovering solution of the present invention. Further, the medium supply/discharge device and the recovering solution supply/discharge device of the present invention comprise a medium container 34, a waste liquid container 36,

pipes

73 and 74, pumps 102 and 103, valves V5, V7, V8, V10 and V11. Further, the stripping liquid supply/discharge device of the present invention comprises a stripping liquid container 35, a waste liquid container 36,

pipes

73 and 74, pumps 102 and 103, valves V6, V7, V8, V10 and V11.

(Effect)

In the present embodiment, after the release solution L is discharged, the cells C are released by the release solution L until remaining, and then the culture medium M is supplied as a recovery solution. Therefore, it is not necessary to separate the cells C from the stripping solution L, and the need for a centrifuge can be eliminated. In addition, since the cells C are separated by the residual stripping solution L in this process, it is not necessary to forcibly separate the cells C adhering to the inner surface of the closed vessel 50 by the force of the fluidized medium M. Further, since the cells C are sufficiently separated from each other by the action of the residual stripping solution L, it is not necessary to break the cell aggregates by a tube pump or the like. Therefore, according to the embodiments of the present invention, damage to the cells C can be suppressed while eliminating the need for a centrifuge.

In the present embodiment, the cell recovery method according to the present invention is performed by the controller 5 appropriately controlling the driving of the pumps 101 to 103 and the opening/closing of the valves V1 to V12. Thus, the cell recovery method according to the present invention can be performed automatically without human intervention.

In the present embodiment, at least two

closed vessels

50 and 60 are connected by a connection path 30. After the cell recovery method according to the present invention is performed in the closed vessel 50, the cell is recovered by introducing a gas (CO)₂) And transferred into a closed vessel 50, and the cell suspension S in the closed vessel 50 is transferred into another closed vessel 60. According to such a configuration, during the subculture in which the cell suspension S is transferred from the closed vessel 50 to the closed vessel 60, the cell suspension S can be transferred without passing through a tube pump or the like. Therefore, damage to the cells C can be suppressed.

(other embodiments)

Modifications to the above-described embodiments will now be described.

In the above embodiments, an example of the operation when the cell recovery method according to the present invention is applied to subculture has been described. Alternatively, the cell recovery method according to the present invention may be applied when the cells are harvested after completion of the culture.

In the above embodiment, a liquid medium is used as the recovering solution of the present invention. However, the type of the recovered liquid is not limited thereto. For example, if the frozen liquid is used as a recovery liquid in a cell recovery operation at the time of harvesting cells, the cells may be cryopreserved after recovery of the cells.

In the above embodiment, each step of the cell recovery method according to the present invention is automatically performed by the controller 5. However, at least a portion of the steps may be performed by an operator.

Description of reference numerals

1: cell culture device

5: controller

30: connection path

50. 60: closed container (Container)

C: cells

M: culture medium (recovery liquid)

L: stripping liquid

S: cell suspension

Claims

1. A cell recovery method for recovering cells cultured in at least one vessel containing a liquid medium and adhering to an inner surface of the vessel, the method comprising performing:

a medium discharging step of discharging the liquid medium from the vessel;

a stripping solution supplying step of supplying a stripping solution for stripping the cells from the inner surface of the container to the container after the liquid medium is discharged;

a stripping solution discharging step of discharging the stripping solution from the container before the cells are completely stripped from the inner surface of the container;

a waiting step of waiting until the cells are peeled off by the residual peeling liquid after the peeling liquid is discharged; and

a recovering solution supplying step of supplying a recovering solution for recovering the cells to the container after the waiting step is completed.

2. A cell culture apparatus configured to perform the cell recovery method of claim 1, comprising:

a medium supply/discharge device configured to supply and discharge the liquid medium to and from the container;

a stripping liquid supply/discharge device configured to supply and discharge the stripping liquid to and from the container;

a recovery liquid supply/discharge device configured to supply the recovery liquid to the container and discharge the recovery liquid from the container; and

a controller for controlling the operation of the electronic device,

wherein the cell recovery method is performed by the controller controlling operations of the medium supply/discharge device, the stripping liquid supply/discharge device, and the recovery liquid supply/discharge device.

3. The cell culture apparatus of claim 2, wherein the at least one container comprises at least two containers, and the at least two containers are connected by a connecting path, and

wherein, after the cell recovery method is performed in one of the at least two containers, the recovery liquid containing the cells in the one of the at least two containers is transferred to the other of the at least two containers by feeding a gas into the one of the at least two containers.